Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 118-131). Also available in electronic version.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Calreticulin (CRT) is a multifunctional Ca2+ dependent chaperone protein, which is localized to the endoplasmic reticulum and plays many important biological roles. In addition to its critical role in cardiovascular development, CRT has been reported to be important for cell migration, adhesion and apoptosis. A few studies have also suggested different roles for exogenous CRT in angiogenesis and tumor growth however no direct evidence for the role of endogenous CRT in these processes is available. To study the in vivo role of CRT in angiogenesis and vascular development, we generated a transgenic mouse overexpressing CRT under the control of the Tie2 promoter (referred to as Tie2-CRT) which is active in both endothelial cells and hematopoietic stem cells (HSCs). The main phenotype of these mice is an increased incidence of lung tumors. These tumors have been characterized according to their histochemical properties as being adenocarcinoma with a Surfactant Protein-C positive (SP-CPos) and Clara Cell Protein negative (CC10Neg) phenotype suggesting an alveolar origin for these tumors. We observed that during the early stages of tumor formation, the lungs show signs of increased inflammation as evidenced by congestion, reddish discoloration and the accumulation of inflammatory cells. We have also identified that the early stage tumors contain cells that express exogenous CRT and HSC markers including CD133, Sca-1, and c-Kit. As the tumor progresses to a fully developed adenocarcinoma, these cells lose the expression of exogenous CRT and HSCs markers and gain an alveolar type II phenotype (SP-CPos). In vitro evaluation of tumor progression using lung tumor cells from Tie2-CRT mice demonstrated a differentiation dependent expression of HSC markers by tumor cells supporting the hypothesis that HSCs might be the cells of origin for the lung tumors observed in Tie2-CRT mice. In summary, the results from this study provide evidence that lung tumors from the Tie2-CRT mice are non-epithelial in origin and that the undifferentiated population of tumor cells have HSC characteristics. After differentiation, these cells lose their stem cell phenotype and acquire an epithelial phenotype. This study is the first to examine the potential link between CRT and lung cancer development.

Calreticulin (CRT) is a multifunctional Ca2+ dependent chaperone protein, which is localized to the endoplasmic reticulum and plays many important biological roles. In addition to its critical role in cardiovascular development, CRT has been reported to be important for cell migration, adhesion and apoptosis. A few studies have also suggested different roles for exogenous CRT in angiogenesis and tumor growth however no direct evidence for the role of endogenous CRT in these processes is available. To study the in vivo role of CRT in angiogenesis and vascular development, we generated a transgenic mouse overexpressing CRT under the control of the Tie2 promoter (referred to as Tie2-CRT) which is active in both endothelial cells and hematopoietic stem cells (HSCs). The main phenotype of these mice is an increased incidence of lung tumors. These tumors have been characterized according to their histochemical properties as being adenocarcinoma with a Surfactant Protein-C positive (SP-CPos) and Clara Cell Protein negative (CC10Neg) phenotype suggesting an alveolar origin for these tumors. We observed that during the early stages of tumor formation, the lungs show signs of increased inflammation as evidenced by congestion, reddish discoloration and the accumulation of inflammatory cells. We have also identified that the early stage tumors contain cells that express exogenous CRT and HSC markers including CD133, Sca-1, and c-Kit. As the tumor progresses to a fully developed adenocarcinoma, these cells lose the expression of exogenous CRT and HSCs markers and gain an alveolar type II phenotype (SP-CPos). In vitro evaluation of tumor progression using lung tumor cells from Tie2-CRT mice demonstrated a differentiation dependent expression of HSC markers by tumor cells supporting the hypothesis that HSCs might be the cells of origin for the lung tumors observed in Tie2-CRT mice. In summary, the results from this study provide evidence that lung tumors from the Tie2-CRT mice are non-epithelial in origin and that the undifferentiated population of tumor cells have HSC characteristics. After differentiation, these cells lose their stem cell phenotype and acquire an epithelial phenotype. This study is the first to examine the potential link between CRT and lung cancer development.

Class I heavy chain (HC) must assemble with β-microglobulin (β2m) and acquire optimal peptide in order to be presented to cytotoxic T cells (CTLs). Calnexin is involved in the initial folding of class I HC and subsequent assembly with β2m. Incorporation of "empty" or suboptimally loaded class I molecules into the multimolecular loading complex is essential for them to acquire optimal peptides. The loading complex consists of several cofactors: TAP, tapasin, ERp57 and calreticulin. The precise role of calnexin and calreticulin in the regulated assembly and peptide loading and the significance of their physical interaction with other cofactors of the loading as well as preloading complex still remains unclear. Using mouse fibroblasts that lack calreticulin, I have studied the role of calreticulin in the assembly and loading of H2-K^b and H2-D^b expressed in these cells. MHC class I molecules in calreticulin-deficient cells are able to assemble with β2m normally, but their subsequent loading with optimal, stabilising peptides is defective despite their ability to interact with the TAP complex. The "empty" or suboptimally loaded class I molecules exit the ER rapidly. Reflecting the loading defect, presentation of endogenously processed antigens by class I molecules in calreticulin-deficient cells is impaired. I have used a human calnexin-deficient cell line CEM.NK^R to study assembly of class I in the absence of calnexin. The results demonstrate that contrary to current understanding, calnexin has an important role in class I HC assembly with 32- microglobulin. The role of heavy chain glycosylation in class I biogenesis is still controversial. My findings suggest asparagine (N)-linked glycosylation of human class I heavy chain at position 86 is optimal and any deviations from "normal" glycosylation results in poor loading with peptides and some defect in the assembly with β2m. Despite affecting the loading function, glycosylation did not have significant effect on presentation of a high affinity binding epitope to HLA-A*0201 specific CTLs. Finally, I show that co-operation from all domains of calreticulin is essential in order to generate a fully functional calreticulin. Interestingly, proline-rich (P) -domain of calreticulin downregulated expression of a number of cellular proteins including MHC class I HC, despite restoring the cytosolic calcium levels in calreticulindeficient cells. The effect of P-domain on class I expression was at the level of transcription.

This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease. / October 2008

Major Histocompatibility Complex (MHC) receptors serve a critical role in self/non-self recognition through the presentation of peptide antigen to circulating T lymphocytes and are also believed to play a role in mate selection. Through the development of antibodies to MHC homologues in trout, this report demonstrates the presence of MHC expression in germ cells, as well as a soluble form in seminal fluid. What role these immune molecules may perform in reproduction and mate selection is discussed. In addition, as ectotherms, fish are often subjected to low temperatures. Previous data indicates that the expression of these genes is abolished by low temperatures. Employing these same antibodies, this report further demonstrates that trout maintain the expression of MH I and its critical light chain component, beta-2-microglobulin when subjected to 2oC for 10 days. Expression of the MH II receptor sub-units however, was sensitive to both confinement stress and low-temperature in vivo, as well as to factors secreted from a known fungal pathogen in cultured macrophage. As the cause of "winter kill", Saprolegniales cultures induced homotypic aggregation and pro-inflammatory gene expression in the macrophage cell line, RTS11 as well as down-regulation of MH II. Though no evidence of fungal toxins was evident, fungal spore size appeared to exceed macrophage phagocytic capabilities. Taken together, such a loss of MH II expression at low temperature may allow for establishment of fungal and bacterial diseases and that upon the return to warmer temperatures, saprolegniales have the ability to maintain MH II down-regulation and evade immune recognition. Concurrent to the study of MH expression, this report includes the first cloning and characterization of calreticulin (CRT) in fish. Like its mammalian homologue and primary chaperone to MHC receptors and other immune proteins, trout CRT appears to be a single copy gene with ubiquitous tissue distribution, displaying anomalous migration as a doublet with relative molecular mass of 60kD. Despite its promoter containing endoplasmic reticulum stress elements (ERSE), trout CRT expression did not increase upon treatment with several calcium homeostasis antagonists. Treatment of peripheral blood leukocytes with phytohemaglutinin did reveal a qualitative increase in cell surface expression, as seen in mammals; however, cellular protein levels did not change, suggesting that, in trout, CRT function may be regulated through cellular sub-localization, rather than through changes in gene expression, as it is in mammals.